Breast cancer is a collection of diseases in which molecular phenotypes can act as both indicators and mediators of therapeutic strategy. Therefore, candidate therapeutics must be assessed in the context of multiple cell lines with known molecular phenotypes. Docosahexaenoic acid (DHA) and curcumin (CCM) are dietary compounds known to antagonize breast cancer cell proliferation. We report that these compounds in combination exert a variable antiproliferative effect across multiple breast cell lines, which is synergistic in SK-BR-3 cells and triggers cell signaling events not predicted by the activity of either compound alone.50. Detection of synergy was performed using nonlinear regression of a sigmoid dose response model and Combination Index approaches. Cell molecular network responses were investigated through whole genome microarray analysis of transcript level changes. Gene expression results were validated by RT-PCR, and western blot analysis was performed for potential signaling mediators. Cellular curcumin uptake, with and without DHA, was analyzed via flow cytometry and HPLC.Dose response curves for CCM and DHA were generated for five breast cell lines. Effects of the DHA+ CCM combination on cell proliferation were evaluated using varying concentrations, at a fixed ratio, of CCM and DHA based on their individual ED- PR- Her2+) relative to the two compounds individually. A whole genome microarray approach was used to investigate changes in gene expression for the synergistic effects of CCM+DHA in SK-BR-3 cells lines. CCM+DHA triggered transcript-level responses, in disease-relevant functional categories, that were largely non-overlapping with changes caused by CCM or DHA individually. Genes involved in cell cycle arrest, apoptosis, inhibition of metastasis, and cell adhesion were upregulated, whereas genes involved in cancer development and progression, metastasis, and cell cycle progression were downregulated. Cellular pools of PPARγ and phospho-p53 were increased by CCM+DHA relative to either compound alone. DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells. The effect was synergistic for SK-BR-3 (ERThe combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon molecular phenotype. DHA enhancement of cellular curcumin uptake is one potential mechanism for observed synergy in SK-BR-3 cells; however, transcriptomic data show that the antiproliferation synergy accompanies many signaling events unique to the combined presence of the two compounds. The pha00 years -38. Prec00 years ,40, duod00 years , stomach00 years , prostat00 years , and bre00 years . CCM has00 years . CCM act00 years . CCM reg00 years ,46-49. C00 years ,50-52.- PR- Her2+), MDA-MB-231 (ER- PR- Her2-), MDA-MB-361 (ER+ PR- Her2+), MCF7 (ER+ PR+ Her2-) and MCF10AT   followed by 5 minutes, 72°C. Primers for PCR reactions are listed in table SK-BR-3 cells were plated in 6 well plates (2.5 × 105/well) in complete medium one day prior to treatment. Following 24 hour treatments with 30 μM DHA, 30 μM CCM, or a mixture containing 12 μM CCM and 18 μM DHA, the cells were lysed with RIPA lysis buffer containing pervanadate (200 μM), NaF (1 mM), diisopropyl fluorophosphates (DIFP), and protease inhibitors . Protein concentrations were normalized using BCA reagent according to the manufacturer's protocol . SDS-PAGE membranes were probed with antibodies to p21, p53 and phospho-p53 , PPARγ and GAPDH .SK-BR-3 cells were seeded into 6 well plates  . A gradient mobile phase composed of 45% acetonitrile (0.1% TFA)-55% water (0.1% TFA) to 100% acetonitrile (0.1% TFA) was used. The flow rate was 1.0 ml/min and the detection wavelengths were 426 nm and 528 nm, respectively, for CCM and rhodamine 6G . The CCM peak was identified by comparing to the reference standard, and the quantification of the CCM was preceded with an external standard curve combined with internal standard technology. The concentration of the CCM was normalized based on the protein concentration of the cell lysate.Quantification of CCM uptake by HPLC was performed to validate the flow cytometry method. Confluent cells in T75 flasks were treated with 20 μM CCM for 24 hours. Following treatment, the cells were washed three times with cold PBS and lysed with RIPA buffer. The lysates were sonicated for 5 seconds. Cell lysate (200 μl) and 20 μl internal standard solution  were added to a pyrex glass tube. Chloroform containing 0.05% BHT (7 ml), 3.5 ml methanol containing 0.05% BHT and 1.7 ml 0.5 M KOAC-HAC (1:1) buffer were added to the tube, vortexed for 1 minute, and centrifuged at 900 × g for 10 min. The chloroform layer was harvested and dried under nitrogen gas flow. The residue was dissolved in 200 μl ethanol and transferred to an HPLC sample vial. CCM uptake was analyzed by a reversed-phase HPLC method using a Shimadzu LC-20AT HPLC system equipped with a multi-wavelength diode array detector (DAD), a SIL-20ACStatistical analysis of data is described earlier for each specific experiment. Otherwise all comparisons were analyzed by ANOVA. P values less than 0.05 were considered significant. Triplicate experiments were repeated at least three times.- PR- Her2+), MDA-MB-231 (ER- PR- Her2-), MDA-MB-361 (ER+ PR- Her2+), MCF7 (ER+ PR+ Her2-) and MCF10AT . ED50 values were determined for DHA and CCM, for each cell line, and presented in Table 50 values) was used to measure the antiproliferation effect for each cell line at different doses (0-100 μM) as described by Tallarida . This indicates that the span of doses where the actual combination of CCM+DHA is effective is much greater than expected. The synergism between DHA and CCM disappeared at higher doses, though a dose-dependent antiproliferative effect was still present. CCM+DHA also affected the MDA-MB-231, MDA-MB-361, MCF7, and MCF10AT cell lines, but subadditive to additive results were observed at all combinations tested  of DHA and CCM using four breast cancer cell lines and one line (MCF10AT) representing 'premalignancy', each with a unique pattern of cellular receptor expression: SK-BR-3 , DHA (30 μM) and CCM + DHA (12 μM + 18 μM). The Agilent whole human genome microarray results for each of the three treatment conditions were compared with transcript levels for SK-BR-3 cells left untreated in growth medium for 24 hours. As described in Methods, Rosetta Resolver was used to assign P values to individual chip elements for differences between untreated and treated cells using an error model-based hypothesis test. All chip elements meeting the criteria of fold-change ≥ 2.0 (p < 0.01), in any experimental replicate, were further filtered to return only transcripts for which at least one chip element responded with 2-fold or greater change, in the same direction, p < 0.01, with no other chip element indicating a significant contradictory directional change, across all three replicates for a treatment. According to these criteria, the numbers of transcripts exhibiting significant change for each treatment are as follows: CCM, 8,817; DHA, 61; CCM+DHA, 1,449 , and annotate genes appearing within this set according to known disease-relevant functions. In addition, CCM+DHA responders below 5-fold were scanned for genes of known relevance to antiproliferation processes, such as caspases. Figure ine panel. TherefoSERPINB5 and CYP1A1/1A2/1B1 are transcripts of special functional interest. SERPINB5 produces a protein also known as maspin, and is thought to be a major downstream effector for the tumor suppression effect of Tamoxifen -68; therAs shown above, our data clearly indicated an induction of genes involved in apoptosis. In order to further validate contribution of apoptosis to the reduction of cell proliferation during CCM+DHA treatment, we used a pan-caspase inhibitor (Z-VAD-FMK) to inhibit initiation of the apoptotic process. Data shown in Figure One common mediator for DHA and CCM is the cell cycle regulator p21. In contrast to our prediction, we found that DHA and CCM alone or in combination do not increase p21 protein expression in SK-BR-3 cells Figure , but canWhile the mechanism of CCM entry into cells is unknown, we have analyzed the differences among four breast cancer cell lines for ability to absorb CCM. We quantified CCM uptake by flow cytometry as CCM is known to fluoresce in the green band ,64. In oin vivo may not be limited to the concentration range previously observed. Careful, tissue-specific pharmacodynamics studies are needed to determine how our cell culture results can translate to a clinical setting, but the practical utility of this compound combination is promising.The idea that changes in diet or diet supplementation may improve the health of cancer patients or enhance the effectiveness of existing treatments is a compelling motivation for exploring the activities of dietary compounds. The translational process for such molecules benefits from a relative lack of toxic side effects and source material that is inexpensive and easily accessible relative to synthetic pharmaceuticals. Human diets can routinely encompass many biologically active small molecules, and evidence for synergy between dietary compounds is emerging ,54,73. I-/Her2+ phenotype is regarded as an aggressive tumorigenic phenotype. Our data suggest that molecular phenotype has specific implications for efficacy, and translation of these compounds into therapeutics will require extensive mechanistic studies. Beyond demonstrating the synergistic effect itself, the importance of this study lies in the demonstration that observed synergy is accompanied by cell signaling changes that are unique to the combination of the two compounds. A network state appears in which the aggregate of protein levels, protein states, and transcript levels are an emergent property of the combined presence of DHA and CCM.As mentioned above, breast cancer is a collection of diseases with multiple phenotypes; therefore, one should not expect that a particular therapeutic agent will control every malignancy subtype. The fact that only the SK-BR-3 cell line was synergistically affected by DHA and CCM suggests specific breast cancer phenotype as an important factor for predicting efficacy. The ERThe results shown in Figures Our microarray data suggest that cell cycle arrest and apoptosis include contributions from other events unique to the CCM+DHA combination upstream, downstream, or independent of PPARγ and phosphorylated p53. Two interaction networks were generated within the MetaCore (GeneGo) network analysis environment to illustrate all kinases documented as activators of p53 and all proteins documented to contribute to upregulation of PPARγ transcription  [in vitro system, has certain limitations, a set of unique genes was identified for the synergistic effect of DHA and CCM. Studies in an in vivo system are currently underway to further validate the findings of the present study.Our data suggest that combination of DHA and curcumin may provide a novel, effective dietary supplemental treatment for some breast cancer patients, with a likely dependency upon molecular phenotype. It is important to stress that the synergistic antiproliferative effects were only observed in the SK-BR-3 cell line. Analysis of other breast cancer cell lines representing the ERlusters) , and theDHA: docosahexaenoic acid; CCM: curcumin; PPAR: peroxisome proliferator-activated receptor; ER: estrogen receptor; PCGL: preselected candidate gene lists; MBGS: Miltenyi Biotec Genomics Services.The authors declare that they have no competing interests.JDA performed experiments, participated in the experimental design, and drafted the manuscript. AAB performed analysis of the microarray results and helped to draft the manuscript. CT participated in the design of experiments, performed statistical analysis and helped to draft the manuscript. KH performed immunoassays and participated in the experimental design. JFV performed immunoassays and HPLC experiments. ZX performed HPLC experiments, analyzed HPLC results, and helped to draft the manuscript. VJD participated in experimental design and helped to draft the manuscript. RAS conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/149/prepubExtracting three-replicate responsive genes from Miltenyi Biotec microarray data. The Excel file "Supplementary Data-1.1.xlsx" demonstrates the first step in logical filtering of preselected candidate gene lists (PCGL) that are provided as pre-processed microarray data by Miltenyi Biotec. Each PCGL is a list of the microarray elements that returned a significant response within one replicate of a treatment (fold change ≥ 2 and P ≤ 0.01). Most transcripts (genes) are represented by more than one microarray element, and these do not always agree with each other, rendering the result for a gene ambiguous. Also, a single microarray address may not return the same result across all three replicates. Therefore, logical filters were implemented as conditional statements in Excel to compile a list, for each treatment, of only those genes that respond significantly in a consistent direction across three treatment replicates. Step one for the Zadd treatment (CCM+DHA) is presented as an example.Click here for fileExtracting identities of genes that did not respond to one treatment but did respond to another treatment. In order to assemble the heatmap in Figure 3, it was necessary for each gene to have a cell value across all three treatments, including those instances in which a particular gene had no response to a given treatment. The Excel file "Supplementary Data-1.2.xlsx" demonstrates, for the Zadd treatment, how to assemble the list of microarray elements that were isolated as three-replicate responders for either of the other two treatments, but that returned no significant response for all three replicates of the Zadd treatment. This list is used in the next step to query the raw data for Zadd in order to collect fold-change values for these elements.Click here for fileExtracting ratio, fold-change and P values for genes that did not respond to one treatment but did respond to another treatment. The microarray element identities compiled in Supplementary Data-1.2 were then used in "Supplementary Data-1.3.xlsx" to query the gene ratio lists  for each replicate of the Zadd treatment. Results for each element are compiled into a single final data set sorted by element ID.Click here for fileMean ratio, fold-change and P values for "ALL not Zadd" microarray element list, checked against three-replicate responders for Zadd. In the Excel file "Supplementary Data 1.4.xlsx", mean ratio, mean fold-change and mean P values are calculated for all microarray elements compiled in Supplementary Data-1.2. These are then sorted by gene ID, and each gene is represented by the microarray element returning the lowest mean P value. This list of genes is then checked against Zadd three-replicate responders in order to catch disagreement among microarray elements.Click here for fileFinal set of Zadd gene responses for all genes that responded to any of the three treatments. This final data set contains treatment/control ratios, fold-change values and P values, from Zadd treatment replicates, for all transcripts that responded ≥ 2-fold, P ≤ 0.01, in any of the three treatments. For any transcript that was not a three-replicate responder in the Zadd treatment, values were derived as described in Supplementary Data 1.1-1.4. The set of values in Excel file "Supplementary Data 1.5.xlsx" served as the source of transcript response values for Zadd in Figure 3. Transcript responses used in Figure 3 for each of the other two treatments, CCM and DHA, were derived using the same workflow.Click here for fileDistribution of fold change by treatment for whole human genome transcript analysis. This figure represents the raw distribution of fold change magnitude for all transcripts that exhibited fold change ≥ 2.0 (p < 0.01) in all three replicates of at least one treatment.Click here for filePossible routes to phosphorylation of p53 and upregulated transcription of PPARγ. p53 and PPARγ were each used as single beginning nodes around which to build one-step expansion networks within MetaCore™ version 6.3 (GeneGo).Click here for fileLiterature annotation for transcript functional categories in Figure Click here for fileData represented by Figure Click here for file


The prognostic implications of P-wave dispersion in patients with a variety of cardiac disease conditions are increasingly being recognised. The relationship between P-wave dispersion and left ventricular function in sickle cell anaemia is unknown.This study was aimed at evaluating the relationship between P-wave dispersion and left ventricular function in adult Nigerian sickle cell anaemia patients.Between February and August 2007, a total of 62 sickle cell anaemia patients  enrolled in the study. These were drawn from patients attending the adult sickle cell clinic of the University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu. An equal number of age- and gender-matched normal subjects served as controls. All the participants were evaluated with electrocardiography and echocardiography. P-wave dispersion was defined as the difference between the maximum and minimum P-wave duration measured in a 12-lead electrocardiogram.r = 0.387; p = 0.031). A comparison of subsets of sickle cell anaemia patients and controls with comparable haematocrit values (30–35%) showed significantly higher P-wave duration and P-wave dispersion in the patients than in the controls. The P-wave duration in patients and controls, respectively, was 111.10 ± 14.53 ms and 89.14 ± 16.45 ms . P-wave dispersion was 64.44 ± 15.86 ms in the patients and 36.43 ± 10.35 ms in the controls . Significant negative correlation was found between P-wave dispersion and left ventricular transmitral E/A ratio .P-wave duration and P-wave dispersion were significantly higher in patients than in controls. Significant correlation was demonstrated between P-wave dispersion and age in the patients (These findings suggest that P-wave dispersion could be useful in the evaluation of sickle cell patients with left ventricular diastolic dysfunction. Further prospective studies are recommended to evaluate its prognostic implication on the long-term disease outcome in sickle cell disease patients. There is increasing recognition of the prognostic implications of the spatial variations of P-wave duration in normal individuals and patients with a variety of cardiac disease states.A cross-sectional study was carried out on 62 sickle cell anaemia patients seen at the adult sickle cell clinic of the University of Nigeria Teaching Hospital (UNTH), Ituku-Ozalla, Enugu from February to August 2007. An equal number of age- and gender-matched normal subjects served as controls. All the participants were evaluated with electrocardiography and echocardiography.Resting 12-lead electrocardiography was performed on all subjects using a Cardioline Ar-600 model electrocardiography machine at a paper speed of 25 mm/s and standardised at 0.1 mV/mm. A single observer analysed the electrocardiogram. The P-wave was measured from the beginning of the P-wave deflection from the isoelectric line to the end of the deflection returning to the isoelectric line. If the beginning or end of the deflection could not be satisfactorily defined, that lead was not used. The difference between the maximum and minimum P-wave duration was taken as the P-wave dispersion.4Echocardiography was done using a Hewlett Packard Sonos 2500 echocardiography machine with 3.7-MHz transducer. The following measurements were taken in the standard positions as recommended by the American Society of Echocardiography:Ethical clearance for the study was obtained from the ethics committee of UNTH, Enugu. Prior informed consent was obtained from all the participants in the study.t-test. In order to examine the effect of anaemia on the variables, the subjects were classified, based on the haematocrit values, into four classes in accordance with the World Health Organisation classification of anaemia as follows: class 1, normal (haematocrit ≥ 36%); class 2, mild anaemia (haematocrit 30–35.9%); class 3, moderate anaemia (haematocrit 21–29.9%); class 4, severe anaemia (haematocrit 18–20.9%).9Data were presented as means ± standard deviation. Comparison of continuous variables between the group of sickle cell disease patients and the control group was made with the independent Student’s post hoc multiple comparison of means using the Tukey’s honestly significant difference test. Intra-class differences in parameters between patients and controls in the same haematocrit class were analysed using the independent Student’s t-test. The relationship between P-wave dispersion and echocardiographic indices of left ventricular function (while controlling for the effect of anaemia) (haematocrit) was examined using the partial correlation analysis.Inter-class differences in clinical, electrocardiographic and echocardiographic parameters in the patients were compared by one-way analysis of variance and p < 0.001, The mean ages of the patients and controls were 28.27 ± 5.58 (range 18–44) and 28.37 ± 5.91 (range 18–45) years, respectively. There were no statistically significant age and gender differences between patients and controls. The patients had statistically significant lower mean values than the controls in the measurement of height, body mass index and body surface area . When subsets of sickle cell anaemia patients and controls with comparable haematocrit values (30–35%) were compared, the patients were found to have significantly higher P-wave duration and dispersion than the controls. The P-wave duration in patients and controls, respectively, was 111.10 ± 14.53 ms and 89.14 ± 16.45 ms . P-wave dispersion was 64.44 ± 15.86 ms in the patients and 36.43 ± 10.35 ms in the controls .P-wave duration and P-wave dispersion were significantly higher in patients than controls In order to evaluate the effect of degree of anaemia on P-wave dispersion in the patients, the electrocardiographic parameters were compared among the haematocrit categories, as shown in r = 0.289; p = 0.023). The correlation was still significant after controlling for the effect of anaemia . Such correlation was not observed in the normal controls .The result of a multivariate Pearson’s correlation analysis of P-wave dispersion and echocardiographic indices of left ventricular function are presented in This study revealed a significant increase in P-wave dispersion in sickle cell anaemia patients. The finding of a negative correlation between P-wave dispersion and left ventricular E/A ratio suggests that left ventricular diastolic function might be deranged in patients with increased P-wave dispersion. Reduced left ventricular relaxation and alteration in left ventricular chamber compliance are the haemodynamic abnormality for left ventricular diastolic dysfunction. Left ventricular hypertrophy, cardiomyopathy, myocardial ischaemia, systemic hypertension and normal aging are recognised causes. In diastolic dysfunction, rapid early filling is decreased, pressure upstream of the left ventricle is increased and atrial systole therefore has a crucial role to play to decrease atrial pressure and resume left ventricular filling.P-wave dispersion has been described as a non-invasive indicator of risk of atrial fibrillation.12P-wave dispersion in sickle cell anaemia positively correlated with patients’ age (duration of illness), suggesting a progressive deterioration with time. Ayetemir and associates,P-wave dispersion was increased in patients with sickle cell anaemia and significantly correlated positively with age and negatively with left ventricular diastolic function. These findings suggest that P-wave dispersion could be useful in the evaluation of sickle cell patients with left ventricular diastolic dysfunction. However, further prospective studies are recommended to evaluate its prognostic implication on the long-term disease outcome in sickle cell disease patients.


Neurokinins (NKs) participate in asthmatic airway inflammation, but the effects of NKs on airway smooth muscle cells (ASMCs) and those of corticosteroids on NKs are unknown.To investigate the effect of budesonide on substance P (NK-1) receptor (NK-1R) expression in the lung and ASMCs, 45 Wistar rats were randomly divided into three groups: control, asthmatic, and budesonide treatment. Aerosolized ovalbumin was used to generate the asthmatic rat model, and budesonide was administered after ovalbumin inhalation. On day 21, bronchial responsiveness tests, bronchoalveolar lavage, and cell counting were conducted. NK-1R protein expression in the lung was investigated by immunohistochemistry and image analysis. Primary rat ASMC cultures were established, and purified ASMCs of the fourth passage were collected for mRNA and protein studies via real-time RT-PCR, immunocytochemistry, and image analysis.NK-1R mRNA and protein expression in the budesonide treatment group rat’s lung and ASMCs were less than that in the asthmatic group but greater than that in the control group.NK-1R is involved in the pathogenesis of asthma and that budesonide may downregulate the expression of NK-1R in the ASMCs and airways of asthmatic rats, which may alleviate neurogenic airway inflammation. Neurokid reflux. In addipatients. Inhaledpatients, and airpatients,7. HowevBordetella pertussis  on day 0 and day 7. Rats in the control group were treated with 1 mL of PBS containing only 200 mg/mL aluminum hydroxide. Two weeks later, the rats were placed in a transparent glass chamber (approximately 20 cm × 20 cm × 20 cm in volume) connected to an ultrasonic nebulizer  and subjected to repeated bronchial allergen challenge via OVA (2%) inhalation for 20 min/day for 6 days. Rats in the control group were challenged with PBS. After OVA inhalation, rats in the budesonide treatment group were given 1 mg of budesonide via inhalation by INQUA NEB plus (PARI) over the course of 5 minutes for 6 days.Forty-five healthy female Wistar rats weighing 150–160 g were purchased from the experimental animal center of China Medical University and divided randomly into three groups: control, asthmatic, and budesonide treatment. All experimental protocols involving animals were approved by the China Medical University Animal Care Committee and complied with the guidelines of the China Council on Animal Care. The modified ovalbumin (OVA)  inhalation method was used to generate the asthmatic rat model as described in detail elsewhere. Briefly2O. Airway resistance was measured by the forced oscillation technique. Five doses of MCh  solution (10–160 μg/mL) in 0.5 mL of PBS were given intermittently via jugular vein injection, each 1 min apart. After each MCh challenge, the respiratory system resistance was recorded by animal pulmonary function analysis software, testing baseline airway resistance and Re, which represents changes in airway responsiveness. When Re reached or exceeded the baseline Re 2 times stop to push MCh.To investigate OVA-induced effects on airway responsiveness, we measured changes in respiratory parameters in response to methacholine (MCh). After the rats were challenged, they were anesthetized with pentobarbital , and the trachea was cannulated with a 14-gauge tube. The rats were quasi-sinusoidally ventilated with a computer-controlled small-animal ventilator  with a tidal volume of 8 mL/kg, set automatically depending on body weight at 90 breaths/min and positive end-expiratory pressure of 3.0 cmHAfter the lung responsiveness measurements, the rats were disconnected from the ventilator and sacrificed via pentobarbital overdose. A catheter was then inserted into the trachea, and BAL was performed. The cell suspension was concentrated by centrifugation , and the cell pellet was resuspended in 1 mL of saline. To perform the differential leukocyte cell count, 0.1 mL of the cell suspension was transferred to a glass slide and stained with Wright-Giemsa stain. A microscope was then used to examine 400 nucleated cells.NK-1R protein expression in the lung was detected by immunohistochemistry. Right middle lobes of the lungs were harvested 24 h after the final OVA challenge and fixed in 4% paraformaldehyde, then imbedded in paraffin. Lung sections of 5 μm were cut and blocked with peroxide and non-immune animal serum and incubated sequentially with primary antibody, biotin-labeled secondary antibody, and streptomycin anti-biotin peroxidase. Finally, the sections were stained with DAB, counterstained with hematoxylin, dehydrated, cleared in xylene, and fixed. Negative staining controls were generated by replacing the primary antibody with PBS. The mean density values of NK-1R protein, indicated by brown staining, were calculated in three selected fields under high magnification (×400) via image analysis. Optical density represented the NK-1R protein content in ASMCs.Twenty-four hours after the final challenge, the rats were sacrificed, and primary ASMCs were cultured according to a previously described method. TracheaThe ASMCs from the different groups were cultured on coverslips, and NK-1R protein expression was detected by immuncytochemistry. After the cells reached confluency, they were fixed in 4% polyphosphate formaldehyde, blocked with peroxide and non-immune animal serum, and incubated sequentially with primary antibody, biotin-labeled secondary antibody, and streptomycin anti-biotin peroxidase. Finally, the ASMCs were stained with DAB, counterstained with hematoxylin, dehydrated, cleared in xylene, and fixed. Negative controls were generated by replacing the primary antibody with PBS. To evaluate positive results, five fields with condensed NK-1R protein expression were selected under low magnification (×100), cell numbers were counted under high magnification (×400), and the mean density values of ASMCs with brown staining were calculated in the five selected fields via image analysis. Optical density represented the NK-1R protein content in ASMCs.′-CGCCGATGTTTCAGTCCATTC-3′, reverse 5′-GACGTATTCAGTCCGTGTTGGTTG-3′; GAPDH forward 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse 5′-ATGGTGGTGAAGACGCCAGT-3′. Gene expression was determined by the 2−ΔΔCT method.To investigate NK-1R mRNA expression in ASMCs from the various groups, real-time RT-PCR was performed as a quantitative analysis. Total RNA was extracted from ASMC cultures using RNAiso™ Plus reagent  and quantified using a spectrophotometer. Following quantification, 2 μg of RNA was reverse-transcribed into cDNA, and real-time quantitative PCR assays were conducted using an ABI PRISM 7500 real-time PCR System  and the SYBR PrimeScript™ RT-PCR kit reagent . The PCR conditions for NK-1R were 45 cycles of denaturation at 95°C for 5 s and annealing and extension at 60°C for 30 s. Target mRNA levels were normalized to those of GAPDH. The following oligonucleotide primers were used: NK-1R forward 5P < 0.05.All experiments were repeated in triplicate. All data were expressed as the mean ± SE and analyzed using SPSS 17. One-way analysis of variance with the SNK test was used to test for significance, which was accepted at in vivo, we measured respiratory parameter changes induced by MCh. Airway responsiveness of rats in the asthmatic group increased after induction by MCh when compared to the control group. However, the airway responsiveness of rats in the budesonide treatment group decreased after induction by MCh when compared with the asthmatic group . Conversely, total cells and eosinophils in the budesonide treatment group were significantly decreased when compared with the asthmatic group (P < 0.05), but did not significantly differ from the control group (P > 0.05)  Table. P < 0.05) but this increase was significantly abrogated by budesonide treatment . Immunohistochemistry was employed to investigate NK-1R protein expression in airways. According to image analysis, the expression of NK-1R protein in the asthmatic group was significantly higher than that in the control group (P < 0.05), but again this level was significantly decreased by budesonide treatment . To investigate NK-1R protein expression in ASMCs, we first studied the purity of ASMCs of the fourth passage via immunocytochemistry. Accordingly, the purity of ASMCs was confirmed to exceed 95% by α-actin staining Figure. Image aP < 0.05) but was significantly lowered by budesonide treatment . Similar to the results observed in all protein expression studies, real-time RT-PCR demonstrated that NK-1R mRNA expression in the asthmatic group was significantly greater than that of the control group  are commonly used therapeutic drugs in the treatment of asthma, and they are generally believed to act through an anti-inflammatory effect that involves decreasing inflammatory factors by inhibiting cytokine production and release. Accordingly, budesonide is an inhaled GC that can inhibit inflammation of the asthmatic airway, but the effect of this drug on neuropeptide-induced inflammation in asthma is unknown.Therefore, in our study, we compared NK-1R expression in the lung and ASMCs obtained from asthmatic rats to those obtained from asthmatic rats treated with budesonide. We found that NK-1R was downregulated in ASMCs, which suggests that budesonide inhibited airway inflammation, at least in part, by downregulating NK-1R expression in ASMCs.et al previously demonstrated the rapid non-genomic effects of GCs on allergic asthmatic reactions in the guinea pig[It has been shown that GCs act through both genomic and non-genomic mechanisms. The genomic effects of GCs are generally mediated by cytosolic receptors that alter the expression of specific genes. Howeverinea pig. Though NK-1R is involved in the pathogenesis of asthma and that budesonide may downregulate the expression of NK-1R in the ASMCs and airways of asthmatic rats, which may alleviate neurogenic airway inflammation.The authors declare that they have no competing interests.ML carried out the ASMC culture, real-time analysis, immunocytochemistry analysis, image analysis, drafted and revised the manuscript. YS participated in the design of the study, analyzed and interoperated the data, and performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/12/79/prepub


PSAP gene that codes for the 65–70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A–D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated. © 2009 Wiley-Liss, Inc.Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the  PSAP gene LacCer, and 50 ng [C17:0]sphingomyelin. The samples were vortexed for a few seconds at 5 min intervals three times. Then 150 µl Milli-Q® water was added and the mixing procedure repeated. After 30 min at room temperature the samples were centrifuged . Each separated lower phase was filtered (PTFE syringe pump filter) and re-washed by mixing with 500 µl Milli-Q® water sulfatide, 35 ng [C17:0]GlcCer, 25 ng [C17:0]Gb3Cer, 50 ng [C17:0]ceramide, 50 ng sphingomyelin (no. 1890), were from Matreya, and [C17:0]ceramide (no. 860517P) from Avanti. Internal standards which were not commercially available, [C17:0]sulfatide and [C17:0]GlcCer, were prepared from lyso-sulfatide  and lyso-GlcCer  by linking the lyso-compounds with C17:0 fatty acid  using enzymatic semisynthesis with sphingolipid ceramide N-deacylase from Pseudomonas sp. TK4  as described Gb3Cer (no. 2923.90), C16:0-DPSAP gene was analyzed for mutations as described previously , NC_000010.9; reference mRNA sequence: GenBank, NM_002778.1; protein sequence: UniProtKB/Swiss-Prot [http://www.expasy.org/sprot/], P07602 [SAP_HUMAN]).Genomic DNA and total mRNA were isolated from the patients' cultured fibroblasts and from the parents' peripheral leukocytes by standard techniques. The Confirming earlier results by A. Bornemann , there was generalized dermal lysosomal storage expressed in the eccrine glands, capillary endothelium , perivasFibroblast homogenates from patient 1 had a partially reduced activity of glucosylceramide β-glucosidase (EC 3.2.1.45), a more markedly reduced activity of galactosylceramide β-galactosidase, but a normal sphingomyelinase (EC 3.1.4.12) activity . The activity of galactosylceramide β-galactosidase was normal in fibroblast homogenates from patient 2 (glucosylceramide β-glucosidase and sphingomyelinase not tested).Thin layer chromatographic analysis of lipids extracted from fibroblasts (corresponding to about 0.5 mg protein) of patient 1 revealed intensely stained spots for ceramide, GlcCer, and LacCer similar to ceramide (derived from loaded [3H]glucosylceramide or [3H]sphingomyelin). For cells from both patient 1 and patient 2, there was a reduced turnover of loaded [3H]globotriaosylceramide and [3H]sulfatide (supporting information S7 may be found in the online version of this article).Metabolic experiments with radioactive sphingolipid substrates (tritium-labeled on their ceramide moieties) loaded onto living fibroblast cultures from patient 1 and patient 2 gave similar results to those described for an earlier pSap-d [Elleder et al., M3 ganglioside (preliminary identification), and sulfatides and dihexosylceramides were also elevated  showed markedly increased levels of Gb3Cer, GlcCer, and Gelevated . SulfatiP < 0.001) to a similar extent to that found in Fabry disease patients (P < 0.001) and the LacCer/digalactosylceramide (P < 0.001), monohexosylceramide , and ceramide (P < 0.001) fractions.In patient 1, the abundance of Gb3Cer was elevated (patients . HoweverP < 0.001) was of a similar magnitude to that found in MLD (P < 0.001) was also remarkable.In patient 2, the elevated concentration of sulfatide  and a premature stop codon. Each of the parents was found to be a carrier for the mutation. While it was possible to amplify the mutant mRNA by RT-PCR in the patient, the same analysis in the parents showed that, in comparison to the wild-type transcript, only trace amounts of the mutant transcript were present.Patient 1 was found to be homozygous for a point mutation, c.1006-2A > G, in the Patient 2 was found to be heterozygous for two mutations. The first mutation, c.577-2A > G, is a splicing mutation located in the acceptor splice site of intron 5. The second mutation is a 2bp deletion, c.828-829delGA, located in exon 8. The latter mutation leads to a frameshift and a premature stop codon. No transcript corresponding to the c.828-829delGA sequence was detected by RT-PCR in the patient. As this mutation leads to a premature stop codon, the most likely explanation for the absence of transcript is nonsense-mediated decay. Two transcripts from the other allele, which carries the c.577-2A > G, were found. Analysis of these indicated that the acceptor splice site in intron 5 was rendered non-functional by the mutation, with either of two different downstream acceptor sites being used instead. In the first transcript a cryptic splice site in exon 6 was used, since the mRNA contained a deletion of 21 bp from exon 6 . In the second transcript the whole of exon 6 was deleted (r.577_720del) and the exon 7 acceptor splice site was used. These in-frame deletions affected only the SapB domain, while the sequence encoding the remaining Saps was intact and in frame. Analysis of the parental genomic DNA showed that the mother was a carrier of c.577-2A > G sequence variant while the father was heterozygous for c.828-829delGA.PSAP gene [Sandhoff et al., Disorders caused by defects in the The diagnosis in the present SapB-d patient was complicated by the early finding of a massive infarction of the left arteria cerebri media, presumably coincidental to the leukodystrophy. However, the finding of high urinary sulfatide eventually led to a molecular diagnosis of SapB-d. This patient was similar to earlier reported patients [e.g., Schlote et al., Urine is an accessible and non-invasive sample that can also be considered to be an “indirect kidney biopsy” for lipid and other analyses, due to the presence of portions of kidney cells [Desnick et al., For patient and control urine samples, the proportion of each of the main sphingolipids relative to the total concentration of these sphingolipids can be calculated without standardization to a reference parameter . In normIn this study we have demonstrated for the first time the use of urinary sphingolipid analysis when diagnosing the rare pSap-d condition. We have also confirmed the usefulness of this procedure when screening for SapB-d and other sphingolipidoses . In partThe c.1006-2A > G splice-site mutation in patient 1 (pSap-d) results in a premature stop codon with, when compared to the wild-type transcript, only traces of the mutant transcript detected by RT-PCR, indicating that it was probably largely removed through nonsense-mediated decay. Consistent with this view, another premature stop codon mutation located downstream of the SapB domain, as in the present patient, also results in an apparently mRNA-negative (pSap-d) allele [Diaz-Font et al., The two mutations carried by patient 2 (SapB-d) affect the fate of the prosaposin transcript in very different ways. A 2 bp deletion (c.828-829delGA) in exon 8 on one allele results in complete absence of the transcript, most likely due to nonsense-mediated decay, so no pSap or Saps would be generated from this allele. On the other allele, the c.577-2A > G splicing mutation leads to the formation of two alternative transcripts, both of which carry an in-frame deletion of a portion of the SapB domain. This mutation exclusively affects the SapB domain and should not result in the deficiency of other Saps. In keeping with this finding, the biochemical and also clinical phenotype in this patient were consistent with an isolated absence of SapB.To date, four different genotypes , including the one for the present patient, have been reported for pSap-d [Schnabel et al., PSAP genotypes may occur which affect the function of more than one Sap domain but less than the whole pSap. In a two-saposin-deficient (Saps C and D inactive) mouse model, the resulting phenotype [Sun et al., PSAP genotypes and phenotypes should contribute to the better understanding of the functions of the pSap/Saps system.However, other mutant


Plasmodium falciparum (PfMSP-119) constitutes the major component on the surface of merozoites and is considered as one of the leading candidates for asexual blood stage vaccines. Because the protein exhibits a level of sequence variation that may compromise the effectiveness of a vaccine, the global sequence diversity of PfMSP-119 has been subjected to extensive research, especially in malaria endemic areas. In Thailand, PfMSP-119 sequences have been derived from a single parasite population in Tak province, located along the Thailand-Myanmar border, since 1995. However, the extent of sequence variation and the spatiotemporal patterns of the MSP-119 haplotypes along the Thai borders with Laos and Cambodia are unknown.The 19-kDa C-terminal region of the merozoite surface protein-1 of the human malaria parasite P. falciparum from five geographically isolated populations along the Thai borders with Myanmar, Laos and Cambodia in three transmission seasons between 2002 and 2008 were collected and culture-adapted. The msp-1 gene block 17 was sequenced and analysed for the allelic diversity, frequency and distribution patterns of PfMSP-119 haplotypes in individual populations. The PfMSP-119 haplotype patterns were then compared between parasite populations to infer the population structure and genetic differentiation of the malaria parasite.Sixty-three isolates of PfMSP-119 were identified. Differences in the prevalence of PfMSP-119 haplotypes were detected in different geographical regions, with the highest levels of genetic diversity being found in the Kanchanaburi and Ranong provinces along the Thailand-Myanmar border and Trat province located at the Thailand-Cambodia border. Despite this variability, the distribution patterns of individual PfMSP-119 haplotypes seemed to be very similar across the country and over the three malarial transmission seasons, suggesting that gene flow may operate between parasite populations circulating in Thailand and the three neighboring countries.Five conserved polymorphic positions, which accounted for five distinct haplotypes, of 19 haplotypes of P. falciparum populations in all endemic populations during three transmission seasons in Thailand were identified, providing basic information on the common haplotypes of MSP-119 that is of use for malaria vaccine development and inferring the population structure of P. falciparum populations in Thailand.The major MSP-1 Plasmodium parasites [Because the clinical symptoms and pathology of malarial infections are caused by the erythrocytic stage cycles of the arasites -3, the darasites . The estarasites , mostly arasites . To datearasites ,8. Despiarasites -11, therPlasmodium falciparum spans ~5 kb in size on chromosome 9 and can be divided into 17 blocks, according to the presence or absence of 9 bp repeats and the type of repeating sequences [msp-1 gene are classified into two allelic types: K1 and MAD20, with the exception of the highly polymorphic block 2 that is represented by at least three allelic types  [83), 30 kDa (MSP-130), 38 kDa (MSP-138) and 42 kDa (MSP-142) [42 fragment is proteolytically processed to form the 33 kDa (MSP-133) and 19 kDa (MSP-119) fragments [19 fragment, containing two epidermal growth factor (EGF)-like domains, remains attached to the parasite’s surface [Components of blood stage malaria vaccines are generally derived from parasite ligands (antigens) expressed on the surface of merozoites, the only extracellular blood stage parasite. Among them is the merozoite surface protein-1 (MSP-1) that is currently used as a major component of anti-blood stage human malaria vaccines ,13. The equences ,15. The nd RO33) . The MSPMSP-142) ,19. Thes surface .msp-1 gene (blocks 2 and 17) have been identified to be prime targets of protective immunity. Previous studies have indicated that natural clinical immunity against blood malaria infections targets the N-terminal fragments encoded by block 2 of msp-1 gene in an allelic-type specific manner [19, encoded by block 17 of the msp-1 gene, could also inhibit erythrocyte invasion by preventing the secondary processing that released MSP-119 from the rest of the MSP-1 complex [19 have been detected in the sera from individuals living in areas that the highly endemic for malaria and were associated with protection from clinical malaria [19-based vaccines are currently in Phase I and Phase II clinical trials in humans and so far have been shown to be safe and immunogenic [Two coding regions of the c manner -24. Natu complex ,25. Inva malaria -33. As munogenic -36.P. falciparum msp-1 gene block 17 sequences has been fairly extensively researched and they have been classified into at least 10 MSP-119 haplotypes in natural populations of P. falciparum[19 haplotypes among different parasite populations revealed that the populations of P. falciparum are genetically diverse [PfMSP-119 haplotypes can also be utilized to infer the population structure of P. falciparum parasites. A recent study indicated the subdivision of two P. falciparum populations in China according to their variations in the distribution patterns of MSP-119 haplotypes, where the parasite populations in Yunnan (South China) appeared to be more closely related to the parasite populations in Southeast Asia (Thailand and Vietnam) than those in Hainan Island located on the South China Sea [Polymorphism in the alciparum-51. Of t diverse , consist diverse ,53. In ahina Sea . The shahina Sea .P. falciparum has been reported in Southeast Asia and has drawn the global attention to the need for improvement in malaria intervention [P. falciparum at the Thailand-Cambodian border in 2008 [P. falciparum isolates at these localities. In Thailand, much of the current knowledge of the msp-1 sequence diversity originated from the analyses of a single parasite population at Mea Sod, Tak province at the Thai-Myanmar border [msp-1 gene block 17 in five geographically isolated populations of P. falciparum in Thailand was performed in order to infer the genetic structure of the parasite populations from the distribution patterns of their MSP-119 haplotypes. The outcome of this work will shed light on the nature and genetic relationship within and between malaria populations and offer a chance to better understand the parasite’s evolution, which will be important for establishing an effective strategy for malaria control and intervention.Recently, the emergence of artemisinin resistance in rvention ,56. Afte in 2008 , artemis in 2008 . Becauser border ,39,51, bP. falciparum were collected from five localities (1-5), each in a different province, close to the borders of Thailand and three neighbouring countries. These were (1) Ubon Ratchathani, located at the Thailand-Laos border, (2) Trat, located at the Thailand-Cambodia border, and (3–5) Ranong, Kanchanaburi and Mae Hong Son, located at the Thailand-Myanmar border. The subjects were confirmed for P. falciparum mono-infection by microscopic examination of Giemsa-stained blood smears. The subjects were recruited in three transmission seasons between: (1) Jun 2002–Oct 2003, (2) Jun 2004–Dec 2005 but also including one subject in 2006 and (3) Jan 2008–Dec 2008  O2 and 90% (v/v) N2. The medium was changed daily, and the parasites were allowed to grow to a parasitaemia level of 5-10% and then harvested prior to genomic DNA preparation.Field isolates of calities -5, each eviously . Of theseviously   saponin solution in phosphate buffered saline  to release the blood stage malaria parasites from the human erythrocytes. The parasite pellets were collected by centrifugation at 10,000 g for 10 min and washed twice in PBS. The parasites were lysed in a lysis solution  sodium dodecyl sulfate, pH 8.0) containing 2 mg/mL proteinase K. The suspension was sequentially treated with an equal volume of phenol (pH 8.0), phenol/chloroform/isoamyl alcohol (25:24:1 (v/v/v), pH 8.0) and chloroform, harvesting the aqueous phase each time. The genomic DNA was then recovered by ethanol precipitation in the presence of 0.3 M sodium acetate and later dissolved in standard TE buffer  and stored at -20°C prior to PCR amplification.Procedures for preparation of the blood stage malaria parasites and genomic DNA were performed as previously described ,61. Briemsp-1 gene block 17 was amplified by the polymerase chain reaction (PCR) with the MM1/17F (5′-TCACAACACCAATGCGTAAAA-3′) and MM1/17R (5′-GAGTATTAATAAGAATGATATTCCTAAG-3′) primer pair, which correspond to nucleotide positions 4,825–4,845 and 5,136–5,109 of the coding sequence of the msp-1 gene of the P. falciparum strain 3D7  of iTaq™-DNA polymerase enzyme in 1X iTaq PCR buffer . Thermal cycling was performed with an optimized profile of an initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 40 s, 53°C for 40 s and 72°C for 40 s, and subsequently a final extension at 72°C for 5 min. PCR products were analysed by standard gel electrophoresis, stained with ethidium bromide and visualized by UV transillumination. All 63 samples generated single amplicons with an expected size of 220 bp, and were subjected to direct Sanger DNA sequencing in both the forward and reverse directions to ensure the accuracy of obtained sequences. Sequencing reactions were performed using the BigDye Terminator v1.1 kit  with an ABI3730 DNA analyser. DNA sequences were manually edited using Bioedit 7.0.0 software and aligned using the MUSCLE Sequence Alignment algorithm in the MEGA 5.2 program [A DNA fragment corresponding to the 1352134, ). The PCPfMSP-119 from the PCR sequenced genomic fragments were aligned with the corresponding sequence of the P. falciparum genome reference strain 3D7 using the MUSCLE Sequence Alignment algorithms as above. The extent of genetic diversity was estimated by the number and ratio of MSP-119 haplotypes prevalent in the populations. Divergences in the distribution pattern of PfMSP-119 haplotypes between two parasite populations was tested for using the Wright’s fixation index (Fst) with Arlequin suite version 3.5 [D*and Fu & Li’s F*indices) implement in the DnaSP 5.1 program [p value was less than 0.05 (p < 0.05).The predicted amino acid sequences of the C-terminal fragment of sion 3.5 . In addi program . ResultsP. falciparum msp-1 gene block 17  at nucleotide positions 4990 G/C, 5132 C/A, 5159 G/A, 5161 G/A and 5206 C/T were detected / Q (CAA), 1691 T (ACA)/K (AAA), 1700 S (AGC)/N (AAC), 1701 G (GGA)/ R (AGA) and 1716 L (CTT)/ F (TTT) . The frequency of amino acid substitutions at each site was E1664Q , T1691K , S1700N , R1701G  and L1716F . The PfMSP-119 fragment contained nine conserved Cys residues. The first amino acid substitution (E1664Q) was located between the second and the third Cys residues in the first putative EGF-like motif, while the other substitutions occurred in the second EGF-like motif of PfMSP-119  and Q/KNG/F, all of which have been reported previously [Nucleotide sequences of be et al.) encodiner et al.) were obeviously -51.PfMSP-119 haplotypes in the five geographical locations of Mae Hong Son (n = 9), Kanchanaburi (n = 15) and Ranong (n = 14), located at the Thailand-Myanmar border, Ubon Ratchathani (n = 12) located at the Thailand-Laos border, and Trat (n = 11), located at the Thailand-Cambodia border  identified. In these three sites, the E/KNG/L, Q/KNG/L and E/TSR/L haplotypes constituted more than 80% of the total haplotypes investigated. Thus, the level of genetic diversity varied according to the geographical location of the parasite populations, which might reflect variation in the transmission intensity and multiplicity of human malaria infections between sampling sites [These results also revealed that there were slight differences in the levels of genetic diversity, based upon the number of s Figure . P. falcng sites .st). In this test, the distribution patterns of PfMSP-119 haplotypes in Tak province (collected in 1995), which is located between Kanchanaburi (the region in which the parasite genetic diversity was highest) and Mae Hong Son (the region in which the parasite genetic diversity was lowest), from two previous studies were also included. One consisted of 8 E/TSR/L, 25 E/KNG/L, 12 Q/KNG/L and 3 Q/KNG/F haplotypes  [st values from the pairs of all the six parasite populations were low and non-significant (p > 0.05). Accordingly, the P. falciparum parasite populations (or at least the PfMSP-119) circulating in this region are genetically homogeneous, suggesting gene flow between allopatric populations of the malaria parasites near the borders of Thailand with Myanmar, Laos and Cambodia.To further determine whether the geographically isolated parasite populations are genetically isolated, pair-wise inter-population comparisons were performed for each parasite population using the Wright’s fixation index  , while tal = 48) . As show19 haplotypes in three transmission seasons is shown in Figure msp-1 sequences analysed, 21 sequences were obtained in a 15-month period between Jun 2002–Oct 2003 and contained three haplotypes ; 25 sequences were obtained in an 18-month period between Jun 2004–Dec 2005 (plus one sample collected in early 2006) and contained all five haplotypes; and 15 sequences were collected in a 12-month period between Jan–Dec 2008. As seen in Figure The prevalence of MSP-1PfMSP-119 haplotypes between years was compared using the Wright’s fixation index (Fst), and this revealed low and non-significant Fst values (p > 0.05)  Table , suggest in 1995 ,39) was P. falciparum in Thailand were found to not be significantly heterogeneous at the PfMSP-119 locus, suggesting that the different PfMSP-119 haplotypes may be maintained by balancing selection. Therefore, intra-population based analyses  were used to evaluate the selection signature on the PfMSP-119 locus. No significant positive D value  was found for the entire block 17 coding region. In addition, although a sliding window analyses of Tajima’s D test, Fu & Li’s D* test and Fu & Li’s F* test revealed positive D values for the EGF-1 and EGF-2 domains, they were not statistically significant  was not analysed because the number of synonymous differences (Ks) was too low (zero). Overall, the polymorphisms at the C-terminal fragment of the msp-1 gene block 17 were less likely to be under strong immune selection, suggesting a functional constraint that limits the allelic diversity of the sequences. Taken together, the polymorphisms detected in PfMSP-119 were neutral and highly conserved in the geographically isolated, but genetically homogenous P. falciparum populations in Thailand.The geographically separate and so potentially isolated populations of PfMSP-1 is considered the major target component for the developmetn of human blood stage malaria vaccines [msp-1 gene block 17 that encodes PfMSP-119 has gained considerable attention in the global malaria research community, leading to the identification of its polymorphism level and haplotypes [PfMSP-119 sequences have been primarily classified into two prototypic allelic groups: (1) the MAD20 prototypic allelic group (E/TSR/L haplotype) and (2) the Wellcome prototype allelic group (Q/KNG/L haplotype) [P. falciparum worldwide [PfMSP-119 sequence diversity in Thailand was previously conducted in a single locality in Tak, located on the western border of Thailand and identified at least five distinct haplotypes  [PfMSP-119 sequence variation is common in other populations of P. falciparum in Thailand or whether the distribution pattern of these haplotypes is conserved. This question formed the basis of the present study, which aimed to screen for polymorphism and evaluate the haplotypes of the msp-1 gene block 17 from different geographical regions in Thailand.The 19-kDa antigenic domain of vaccines . Therefoplotypes -51. PfMSplotype) . In addiorldwide -51. Thesorldwide . The anaQ/KNG/F) . Howevermsp-1 gene block 17 was performed on 61 Thai P. falciparum isolates originating from five endemic areas; Mea Hong Son, Kanchanaburi and Ranong, along the borders of Thailand and Myanmar, Trat at the border of Thailand and Cambodia, and Ubon Ratchathani, at the border of Thailand and Laos. These areas were among the top 15 provinces with the highest numbers of P. falciparum malaria cases in Thailand [PfMSP-119 sequences  previously identified in Tak [msp-1 gene block 17 in Thai P. falciparum populations was limited.Nucleotide sequence analysis of the Thailand . The fivd in Tak ,39,51 and in Tak ,40-50, wmsp-1 sequences in Thai populations varied according to the geographical locations. The lowest genetic diversity was detected in P. falciparum populations in Mae Hong Son and Ubon Ratchathani, with only three PfMSP-119 haplotypes of being found, compared to four and five in Kanchanaburi and Ranong, respectively. Variation in the levels of genetic diversity in geographically isolated populations may be due to variation in the multiplicity of infection, malaria incidence, transmission intensity and levels of gene flow between the parasite populations [P. falciparum were highly prevalent at the Thailand-Myanmar border region in Kanchanaburi compared to at the Thailand-Laos borders [msp-1 alleles in the mosquito and subsequently a high diversity of msp-1 would be maintained in Thai populations [The levels of genetic diversity of the ulations ,66. Malaulations . In addi borders . Elevate19 haplotypes in Thai populations in the present study were compared with results from a previous study based upon microsatellite typing [When the diversity and distribution patterns of MSP-1e typing , no genePfMSP-119 in populations in Thailand, as detected using F-statistics (Fst), revealed no significant differences between the P. falciparum populations located at the Thai borders with Myanmar, Laos and Cambodia. Accordingly, there may be no barriers that limit gene flow between geographically isolated P. falciparum populations in Thailand. This would likely reflect that there are numerous host (human) movements within and into Thailand from neighboring countries from immigrants who live across the borders [P. falciparum occurred around the Thai-Cambodia border in 2009 and now has recently been found at the Thai-Myanmar border in 2011. The results from this study suggest that it is likely that the artemisinin resistant strains of P. falciparum could also spread to other regions of Thailand.The level of genetic differentiation of  borders ,70. For  borders . A cross borders . The maj19 haplotypes in four transmission seasons (from 1995 to 2008) revealed that the overall frequency of individual PfMSP-119 haplotypes was relatively stable, although the two major haplotypes (E/KNG/L and Q/KNG/L) inversely fluctuated over time. This result was also seen in a previous longitudinal survey in Mali [PfMSP-119 haplotypes in endemic regions worldwide indicated a dramatic distribution pattern and frequency of PfMSP-119 haplotypes. The high prevalence of the E/KNG/L and Q/KNG/L haplotypes reported in Thailand has also been reported in the Yunan and Hainan provinces in China and Vietnam [P. falciparum populations in the Greater Mekong Sub-region (GMS)  could be genetically related. Whether the parasite populations in GMS are genetically homogeneous will need to be evaluated by large-scale genotyping approaches using genome-wide genetic markers  appeared to be the dominant haplotypes in other regions, such as South Asia (India and Iran), South America (Brazil and Peru), Vanatu and the Solomon Islands [PfMSP-119 haplotypes, this study could help inform choices about which PfMSP-119 haplotypes to formulate future vaccines from and to allow a more accurate interpretation of the efficacy of current formulations of PfMSP-119 based vaccines being tested in clinical trials. Currently, MSP-1 sequences from P. falciparum K1 (Q/KNG/L) are being formulated as components of the multivalent blood stage vaccine PfCP-2.9 [PfCP-2.9 vaccine would be advantageous for populations in the GMS and in Africa, but not in South America and the Asian Pacific Islands.The longitudinal survey of MSP-1 in Mali . Compari Vietnam ,46, suggNPs, see ). Similaand Mali -44. In c Islands -41,50. BPfCP-2.9 . If the 19 sequences may be due to positive selection or balancing selection, as has been demonstrated for other regions of MSP-1 [D* test and Fu & Li’s F* test all failed to reveal any statistically significant evidence for strong positive or balancing selection of the C-terminal fragments of PfMSP-119 in these Thai samples. The most probable explanation for the limited diversity in PfMSP-119 is the functional constraint of the protein. MSP-1 is considered to play an important role in erythrocyte entry by the merozoite [PfMSP-1 to PfMSP-119 is a prerequisite for merozoite invasion [PfMSP-119 fragment possesses different epitopes and variation in the motifs alters the conformational structure markedly [19 amino acid sequences.It is possible that the limited allelic diversity in the MSP-1of MSP-1 . Howevererozoite . Secondainvasion . The PfMmarkedly ,75. It mmarkedly . Thus, sPfMSP-119 polymorphisms in natural populations of P. falciparum in Thailand. The data supports the view that P. falciparum populations in the GMS region are genetically diverse. Analysis of the spatiotemporal patterns of MSP-1 haplotypes indicated that E/KNG/L and Q/KNG/L were the major haplotypes in Thailand, which were also highly prevalent in populations from other Southeast Asian countries, China and Africa. This study provides the basis for the accurate measurement and interpretation of the population structure and dynamics of malaria parasites that is critical for monitoring the population responses to MSP-1 based vaccines in clinical trials.This study extends the understanding and current knowledge of the variation and prevalence of The authors declare that they have no competing interests.PS carried out the molecular genetic work, interpretation of data and drafted the manuscript. SP performed data analysis and wrote the manuscript. NS and AS were responsible for parasite cultivation. MK and RB carried out the statistical analysis and guidance for data interpretation. PH provided conceptual framework for the project, guidance for the interpretation of data, participated in the manuscript preparation, revision and coordination. All authors read and approved the final manuscript.The origin and date of collection of the human malaria parasite Plasmodium falciparum used in the present study. Asterisks (*) indicate samples collected between 2002 and 2006 that were previously genotyped by 12 microsatellite loci and confirmed to be independent clones [t clones .Click here for fileNucleotide sequence alignment of the Plasmodium falciparum merozoite surface protein-1 (msp-1) gene block 17. Data shows the representative five variants  of the 61 samples analysed. The nucleotide positions are presented on top of the P. falciparum 3D7 sequence . Nucleo1352134; . AsterisClick here for file


S. sq VEGFR mRNA was expressed at highest levels in circulatory system-containing tissues, suggesting that S. sq VEGFR plays an important role in the formation or maintenance of circulatory system in Tunicates, Halocynthia roretzi.The vascular endothelial growth factor (VEGF)-VEGF Receptor (VEGFR) system is an important pathway for regulation of angiogenesis. However, its evolutionary development, particularly the step from invertebrates to vertebrates, is still largely unknown. Here, we molecularly cloned the VEGFR-like gene from Halocynthia roretzi, a species belonging to the Tunicata, the chordate subphylum recently considered the sister group of vertebrates. The cDNA encoded a homolog of human VEGFR, including the transmembrane domain, and the tyrosine kinase domain with a kinase-insert region, which was designated S. sq VEGFR (GenBank AB374180). Similar to Tunicates including ascidians in the phylogenetic tree, the Amphioxus, another chordate, is located close to vertebrates. However, S. sq VEGFR has a higher homology than the Amphioxus VEGFR-like molecule (GenBank AB025557) to human VEGFR in the kinase domain-2 region. The  VEGFVascular endothelial growth factorVEGFRVascular endothelial growth factor receptorS. sq.Sea squirtB. schlosseriBotryllus schlosseriH. roretziHalocynthia roretziC. intestinalisCiona intestinalisCephalochordates (Amphioxus), and Tunicates, which include the ascidians such as Halocynthia roretzi (H. roretzi)  and Ciona intestinalis  have been extensively studied on their development, morphogenesis and phylogeny. Particularly, B. schlosseri is unique in terms of their asexual growth of zooid and their circulatory system . Ho. HoH. ro, 67.1%)  than theThe “Y-K-D/N-P-D-Y” amino acid residue at KD2 of human VEGFR (residue 1048–1053 in human VEGFR1) is highly conserved among VEGFRs in vertebrates. VEGFR1 in mammals and birds have the sequence “Y-K-N-P-D-Y” whereas VEGFR2 and VEGFR3 in essentially all the vertebrates reported so far have “Y-K-D-P-D-Y”. The last “Y” corresponds to the 416-tyrosine in c-Src tyrosine kinase which is critical for activation of Src-kinase . PDGFR fB. schlosseri and C. intestinalis, with minor modifications , followed by the intestine and muscle, and lower in the pharynx and gill  suggests an intimate relationship between S. sq VEGFR mRNA and the circulatory system in this animal. Western blot analysis also revealed that the S. sq VEGFR is highly expressed in the stomach (including heart), and then intestine and significantly less abundant in gill, and pharynx . This sequence shows a high homology at amino acid levels in tyrosine kinase domain (KD1 and KD2), except for the kinase insert region, with that of the sequence S. sq VEGFR (H. roretzi)  . Howeveret al. isolated a B. schlosseri cDNA encoding VEGFR-related molecule [VEGFR mRNA is expressed in the epithelial cells covering the ampullae, and suppression of the gene expression via siRNA or human VEGFR-kinase inhibitor significantly attenuated the angiogenesis occurred after ampullaectomy and disorganized the whole morphology. These results suggest that VEGFR function is involved in a unique angiogenic process and morphogenesis in B. schlosseri.More recently, Tiozzo molecule . By usinIn this study, we showed a higher homology of S. sq VEGFR (KD2 region) to human VEGFR compared to that of Amphioxus VEGFR to humans. This might indicate a strong phylogenetic affinity between tunicates and vertebrates. However, the information on the homology derived from a single gene product such as VEGFR is limited, and it could be modified with the structure of interacting molecule(s). Thus, the views on the relationships between the chordates should be carefully studied both at genome-wide level and each gene product level ,14,34–37Both VEGFs and PDGFs and their receptors have been implicated in mammalian vascular development, and the prototype VEGF (VEGF-A) also regulates the development of hematopoietic cells. A VEGFR was found in Drosophila; however, there is no blood vessel structure in this animal. The VEGFR in Drosophila was shown to regulate the migration of border-cells, hemocytes, and thoracic epithelial cells ,38,39.Halocynthia roretzi (S. sq VEGFR) should shed more light on the function of VEGFR in angiogenesis.To the authors’ knowledge, this is the first report for a VEGFR in Halocynthia roretzi that express significantly in the tissues containing hearts and circulatory system. However, it is not confirmed yet whether the S. sq VEGFR is expressed in the epithelial cells surrounding vessel-like structure and hematopoietic cells in this animal. Further analysis of this molecule in H. roretzi was selected as the ascidian in our experiment. After the surface of each individual was washed with Ca2+ and Mg2+-free artificial sea water , 5 mM EDTA-2Na, and 6 mM NaHCO3), the tunicate was removed and the exposed inner walls were opened. We mainly picked up heart (with part of stomach), intestine, gill, and pharynx for histological analysis.H. roretzi. Fresh tissue from different parts of H. roretzi was minced on ice and then homogenized at room temperature with 1 mL of denaturing solution  in a glass-Teflon.The AGPC method (Chomczynski and Sacchi 1987)  was usedFirst-Strand cDNA was synthesized according to the protocol of the SMART™ cDNA Library Construction Kit . Next, we amplified the cDNA by long-distance PCR (LD PCR). Then we purified the PCR product according to the protocol of the Qiaquick PCR Purification Kit . Finally, cDNA size fractionation was carried out using CHROMA SPIN-400 columns according to the manufacturer’s protocol.VEGFR genes highly conserved among various species.Degenerated oligonucleotide primers  were designed based on the sequences of -actin genes highly conserved among various species. Rapid amplification of cDNA ends of the 5′-or 3′-end was performed with these degenerate primers and anchor primers corresponding to the anchor sequence combined with the 3′-end of the oligo(dT) primer or the sequence involved in the 5′-end of the SMART IV oligonucleotide. The nucleotide sequences were determined using a BigDye Terminator cycle sequencing kit and an ABI310 automated DNA sequencer . To further extend the S. sq VEGFR sequence, we generated several primers based on partial sequences of S. sq VEGFR determined as the original, in both the 5′ and 3′ direction.Degenerated oligonucleotide primers  were designed based on the sequence of β4, and 15 pmol of each primer. PCR was performed with one cycle of incubation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 15 s and 50 °C for 30 s, with a final cycle at 68 °C for 5 min. The PCR products were separated by electrophoresis in a 1.5% agarose gel and then purified by gel extraction. Sequencing was performed with a PE-Biosystems BigDye terminator kit and an ABI Prism 377 XL sequenator.PCR was performed in a 20 μL reaction mixture containing 1 μL of cDNA, 5 μL of 10+PCR buffer, 0.2 mM of each dNTP, 1 mM of MgSOH. roretzi.HaeIII, Saw3AI, HinfI, and SspI as restriction enzymes have been used for checking the existence of Eph receptors as a positive control in Tissue samples were fixed in 4% paraformaldehyde (PFA). For Hematoxylin and Eosin staining, paraffin-embedded specimens were sectioned at a thickness of 5 μm and stained with hematoxylin and eosin using a standard protocol.H. roretzi tissues  using the AGPC method [-actin gene in the ascidian tissues, as the positive control. After finding the sequence of β-actin in H. roretzi, we generated a pair of primers from the β-actin sequence for RT-PCR .Total RNA samples were exacted from the different C method . ReverseTo generate antibody against the S. sq VEGFR protein, the 20-amino-acid peptide “CEGANSMKRGKRSDEDKDPD” which corresponds to a part of the kinase-insert region within the tyrosine kinase domain, was synthesized and conjugated with the KLH carrier. Rabbits were immunized several times with the conjugated protein, and antiserum was obtained.Proteins separated by SDS-PAGE were transferred to Immobilon P membrane. The membrane was blocked with 5% BSA in PBST buffer and incubated with purified primary antibody for 2 h. Corresponding horseradish peroxidase-conjugated secondary antibodies were used for detection of the primary antibody with chemiluminescent reagent.DNA Data Base Accession number for S. sq VEGFR: GenBank AB374180.Halocynthia roretzi, one of three basal chordates. S. sq VEGFR has a higher homology than the Amphioxus VEGFR-like molecule to human VEGFR. The S. sq VEGFR mRNA was expressed at higher levels in heart-localized tissue (stomach) and circulatory system-containing tissue (intestine), suggesting that S. sq VEGFR plays an important role in the formation or maintenance of circulatory system in tunicates, H. roretzi.For the first time, we molecularly cloned the VEGFR-like gene in


Currently, no standardized method is available to predict success rate after percutaneous nephrolithotomy. We devised and validated the Seoul National University Renal Stone Complexity (S-ReSC) scoring system for predicting the stone-free rate after single-tract percutaneous nephrolithotomy (sPCNL).The data of 155 consecutive patients who underwent sPCNL were retrospectively analyzed. Preoperative computed tomography images were reviewed. The S-ReSC score was assigned from 1 to 9 based on the number of sites involved in the renal pelvis (#1), superior and inferior major calyceal groups (#2–3), and anterior and posterior minor calyceal groups of the superior (#4–5), middle (#6–7), and inferior calyx (#8–9). The inter- and intra-observer agreements were accessed using the weighted kappa (κ). The stone-free rate and complication rate were evaluated according to the S-ReSC score. The predictive accuracy of the S-ReSC score was assessed using the area under the receiver operating characteristic curve (AUC).The overall SFR was 72.3%. The mean S-ReSC score was 3.15±2.1. The weighted kappas for the inter- and intra-observer agreements were 0.832 and 0.982, respectively. The SFRs in low (1 and 2), medium (3 and 4), and high (5 or higher) S-ReSC scores were 96.0%, 69.0%, and 28.9%, respectively (p<0.001). The predictive accuracy was very high (AUC 0.860). After adjusting for other variables, the S-ReSC score was still a significant predictor of the SFR by multiple logistic regression. The complication rates were increased to low (18.7%), medium (28.6%), and high (34.2%) (p = 0.166).The S-ReSC scoring system is easy to use and reproducible. This score accurately predicts the stone-free rate after sPCNL. Furthermore, this score represents the complexity of surgery. The incidence of urolithiasis is approximately 5% to 10% in the general population, and 30% of the surgical workload is related to urinary stone treatment in an active urologic department Our experience performing PCNL led to the hypothesis that the distributional complexity of stones is more associated with the SFR than the number or size of stones. Thus, we devised the Seoul National University Renal Stone Complexity (S-ReSC) scoring system to predict the SFR after PCNL. This scoring system is simply calculated by counting the number of sites involved among 9 pre-determined locations in the pelvicalyceal system. We evaluated the ability of the S-ReSC scoring system to predict the SFR and represent the surgical difficulty of single-tract PCNL (sPCNL).This study was approved by the Institutional Review Board (IRB) of Seoul National University Bundang Hospital . The approval number is B-1211/178-109. We were given exemption from getting informed consents by the IRB. It was a retrospective study. And personal identifiers were all removed and the data were anonymously analyzed.A total of 155 consecutive sPCNL cases were included from January 2004 through July 2012 at Seoul National University Bundang Hospital. Percutaneous renal access was routinely obtained by 2 experienced uro-radiologists 1 day before or on the operative day. If the patient previously had a percutaneous nephrostomy, this nephrostomy was used as an access. sPCNL was performed in a prone position by 1 of 4 faculty professors. A rigid nephroscope was used in combination with a ballistic lithotripter, stone forceps, and a suction tube. If needed, a flexible nephroscope and/or ureteroscope were also used for collecting systems that were inaccessible with a rigid nephroscope. In this setting, a Holmium laser and stone basket were used. Temporary drainage was usually maintained with a 14-F nephrostomy catheter.All patients were evaluated with pre- and post-operative computed tomography (CT). The evaluated preoperative stone parameters included the number, largest diameter, total stone volume, renometry , average Hounsfield units, and degree of hydronephrosis . A complete staghorn stone was defined as a renal pelvic calculi extending into all major calyceal groups filling at least 80% of the renal collecting system, and a partial staghorn stone was defined as a renal pelvic calculi extending into at least two calyceal groups. Stone volume was calculated by length×width×depth×π×0.52. The total stone volume was the sum of all stone volumes. The average Hounsfield unit was measured using the elliptical region of interest incorporated into the largest stone area in a non-contrast axial image The S-ReSC score was calculated by counting the number of sites involved, regardless of the size and number of stones. The sites were as follows; the renal pelvis (#1), superior and inferior major calyceal groups  (#2–3), and anterior and posterior minor calyceal groups of the superior (#4–5), middle (#6–7), and inferior calyx (#8–9). The anterior and posterior division was simply divided using the frontal plane of the kidney. Each minor calyceal group could have more than one calyx. Minor anatomical variants in the minor calyceal system were simplified to provide simple and quick scoring and augment reproducibility. One point was given for each location, resulting in a maximum of 9 points in total .Univariate logistic regression analyses were performed to verify the effect of stone existence at each site.Initially, 1 junior faculty member appraised the S-ReSC score. This score set was used for the other statistical analyses. To test intra-observer agreement, this faculty member rescored the patients 1 month later and was blinded to the first reading. To assess inter-observer agreement, one senior resident independently scored the S-ReSC. Inter- and intra-observer agreements were conservatively evaluated using the weighted kappa (κ) with the squared method.The SFRs were examined according to the S-ReSC scores. To evaluate the predictive accuracy, the area under the receiver operating characteristic curve (AUC) was used. Next, the predicted probability versus the actual SFR was compared. This examination was performed by calibration plotting using 200 bootstrap resamples. To test the clinical utility of the S-ReSC score, a decision curve analysis (DCA) was constructed. The AUC value only presents the predictive accuracy, while a DCA depicts the potential net benefit of the predictive model at each threshold probability using a graph We also performed univariate logistic regression analyses to predict the stone-free. The assessed variables were the S-ReSC score, age, sex, laterality, body mass index, previous treatment, stone number , largest diameter, total stone volume, renometry, average Hounsfield units, and degree of hydronephrosis. To adjust for other variables, the S-ReSC score and all significant variables (p<0.05) were included in a multiple logistic regression analysis.To evaluate the correlation between the S-ReSC score and the difficulty and complexity of the surgery, we compared the operative time, hemoglobin drop (preoperative hemoglobin – immediate postoperative hemoglobin), and complication rate. Complications were assessed and scored according to the modified Clavien classification http://www.r-progect.org/). Calibration plot and decision curve were also generated using this software package.The data are expressed as the mean ± standard deviation of the mean. To compare mean values, one-way analysis of variance (ANOVA) test was used. When other statistical test was used, we indicated the method with the results. A p-value <0.05 was considered to be statistically significant. All statistical analyses were performed using R for Windows, version 2.15.2 (The basic characteristics of the patients are shown in The mean S-ReSC score was 3.15±2.12. The S-ReSC scores of complete (7.16±1.36) and partial staghorn stones (4.17±1.14) were significantly higher than the other stone types . The effect of stone existence in the anterior calyces on the SFR was similar to posterior calyces .The weighted kappas for inter- and intra-observer agreement were 0.832  and 0.982 (95% CI 0.971–0.992), respectively, indicating that the scale could be interpreted as having “very good reliability”. The senior resident (2.79±2.02) tended to score lower than the junior faculty member . However, the intra-observer agreement was almost perfect. Major disagreement occurred for S-ReSC scores of 4 or higher.The SFRs according to the S-ReSC scores are shown in The S-ReSC score, stone number, largest diameter, total stone volume, renometry, average Hounsfield units, and degree of hydronephrosis were significantly associated with the stone-free. Even after adjusting for all other significant variables, the S-ReSC score was strongly correlated with the stone-free after sPCNL .The higher S-ReSC score group had a significantly longer operative time  and larger hemoglobin drop . The complication rates showed an increasing trend in order to S-ReSC score groups . A total of 53 complications occurred in 39 (25.2%) patients. Most complications were mild and transient, but 7 (4.5%) and 4 (2.6%) patients experienced grade IIIA and IIIB complications, respectively .Understanding the urinary collecting system is very important to reliably perform endourologic procedures and uro-radiologic evaluations. Sampaio and colleges described the pelvicalyceal morphologic classification in an analysis of 140 3-dimensional polyester resin corrosion endocasts obtained from 70 fresh cadavers, and they proposed 2 major groups If a surgeon can access a certain pelvicalyceal site, they may be able to remove nearly all of the stones, regardless of their number or size in that location. The number of stones in different sites is clearly associated with the S-ReSC score. The stone burden is commonly expressed as the largest diameter or the estimated total stone volume 3 and <5% of stones in unfavorable calyces; type 2, between type 1 and 3; and type 3, total stone volume >20,000 mm3 and >10% of stones in unfavorable calyces. An unfavorable calyx was defined as a calyx with an acute angle (no specific cut-off point was given) and an infundibular width of less than 8 mm. This classification is based on an exact measurement, but it is so complex and requires special software. Therefore, it is difficult to use in daily practice in most urological clinics. By contrast, the S-ReSC scoring system does not require additional software and is easy to use, requiring approximately 15 seconds to score in our experience.Several groups have attempted to identify significant predictors of the stone-free after PCNL The ideal prediction model in medicine should be generally accurate, well calibrated, simple, reproducible, and clinically meaningful There are several limitations to the present study. This was a single-center study. Additionally, this is only a proposal of a predictive method for the SFR after PCNL and a descriptive method for presenting the complexity of renal stones. Thus, a larger scale multi-center study should be performed to validate our results. In our institution, experienced uro-radiologists obtain percutaneous renal access. If an urologist accesses the kidney, the stone-free probability and complication rate could be different. Additionally, if multi-tract or modified PCNL, such as supine PCNL, is performed, the results of the scoring system should be first confirmed.In summary, the S-ReSC scoring system is easy to use and reproducible. This score accurately predicts the SFR after sPCNL. Furthermore, this score represents the complexity of surgery. Thus, the S-ReSC scoring system can be used as a predictive method to estimate the SFR after sPCNL and as a method for describing the complexity of renal stones.